The polysaccharide capsule of Streptococcus pneumoniae is the major virulence determinant of this organism. Of 84 different capsular types, 20 cause greater than 90% of all pneumococcal infections. The relative importance of capsular type in the pathogenesis of S. pneumoniae is not known. Epidemiologic studies cannot distinguish effects due to capsular type from those due to non-capsular determinants. Comparisons of the virulence of isogenic strains of different capsular type have not been conducted. In the proposed studies, isogenic strains will be conducted using isolated restriction fragments to transform capsular genes. Because the fragments used will carry only the capsular genes and a minimum amount of capsular genes. Because the fragments used will carry only the capsular genes and a minimum amount of flanking DNA, the transformants will differ from the recipient parent strain solely in the type of polysaccharide capsule produced. Because only the capsule type will be changed, the relative contributions of capsular type and somatic antigens can be assessed. This will be done by determining virulence of the isogenic strains in mice, and by examining the ability of antibodies to pneumococcal cell wall determinants to protect the mice against infection. Previous studies have shown that antibodies to phosphocholine (PC) and pneumococcal surface protein A (PspA) protect mice against infection with some S. pneumoniae isolates but not others. Protection studies using the isogenic strains will shown whether the determining factor in the protective abilities of these antibodies is the type-specific capsule of the organism. Genetic analyses of S. pneumonia capsules predate those of other bacteria and suggest that the capsular genes are genetically linked in those chromosome; that capsular genes of different types occupy identical sites in the chromosome; that the genes are exchanged as a unit during transformation; and that unlinked regulatory genes are also involved. These early results have not been pursued with modern genetic molecular techniques, and knowledge of capsule genetics has hardly advanced in the last 15 years. In the proposed studies, transposon mutagenesis with Tn916 will be used to generate capsule-negative mutants. The chromosomal locations of the insertions will be determined, and the ability of Tn916 to cotransform with the capsule-negative phenotype will be tested. These experiments should provide direct evidence for the previously hypothesized mechanisms. They will also permit more extensive molecular studies, including cloning of the capsular genes. From this will come information concerning gene products regulation of capsular gene expression, and more insight into the mechanism of capsular gene exchange. The cloned genes will be used to confirm and extend the virulence data obtained with the original isogenic strains. If differences in capsular type cause differences in virulence, an understanding of the genes involved in capsule synthesis will eventually allow the construction of understanding of the genes involved in capsule synthesis will eventually allow the construction of recombinant strains to identify the structural features causing the differences in virulence.